Ncr nu nu

Mice were housed and treated according to guidelines established by the Institutional Animal Care and Use Committee of Rutgers University, who also approved all mouse experiments (animal protocol PROTO99900044, approval date 10/16/2019 – 10/15/2022). Nude mice (NCR nu/nu) were purchased from Taconic Biosciences. Xenograft tumors were generated from the stable tumor cell lines H1299-V272M and H1299-E285K (1 × 10⁷ cells/tumor site/mouse). Tumor dimensions were measured every 1–4 d and their volumes (V) were calculated by using the formula: V = (length × width² × π)/6. Tumors were allowed to grow to 50 mm³ at which point the mice were randomly allocated to treatment and control groups and ZMC1 or vehicle was administered.

Congenitally athymic nude mice (NCr-nu/nu) (6 to 8 wk age) were bred in-house or purchased from Taconic Biosciences. Mice were housed under pathogen-free conditions. All animal experiments were performed with the approval from the University of Nebraska Medical Center Institutional Animal Care and Use Committee.

Human cell lines were transplanted into the testes of busulfan-treated, immune-deficient nude mice (NCr nu/nu; Taconic) as previously described for primate and human spermatogonia (Ramathal et al., 2014, Hermann et al., 2010). Two clonal cell lines each of the iAZFΔa-DDX3Y-Flag-mCherry, or the control line iAZFΔa-mCherry were transplanted. Briefly, immunodeficient nude mice were treated with a single dose of busulfan (40 mg/kg, Sigma) at 6 weeks of age to eliminate endogenous spermatogenesis. Xenotransplantation was then performed five weeks after busulfan treatment by injecting 7–8 μl cell suspensions containing 500,000–600,000 cells total (either iAZFΔa-DDX3Y-Flag-mCherry or iAZFΔa-mCherry lines), or a mixture of equal parts of both lines. Three mice were xenotransplanted in total per cell line (i.e. six testes in total per cell line). All cell suspensions contained 10% trypan blue (Thermo Fisher Scientific) and were directly injected into the seminiferous tubules of each recipient testis via cannulation of the efferent ducts. Eight weeks after transplantation, recipient mouse testes were harvested for donor cell isolation, whole-mount immunostaining and whole testes immunohistochemical analyses.